(A) Schematic showing sites of viral injection at P1. (B,C) Example sagittal and coronal sections of P6 and P5 mouse brains, respectively, showing widespread expression of GCaMP6s across the cortex and several other brain regions including hippocampus, midbrain, and thalamus. Scale bar = 2 mm. Exposure time: 1000 ms. Minimum-Maximum display range in ImageJ (Unsigned 16-bit range): (B) 73–1200; (C) 0–4095. (D–E) Example sagittal and coronal sections at P21 showing that expression of GCaMP6s at P21 is brighter than at earlier ages. Exposure time: 1000 ms. Minimum-Maximum display range in ImageJ (Unsigned 16-bit range)=0–4095. (F) Sagittal section of P45 brain injected at P1 with 4 μL of AAV9 (1 × 1013 vg/mL. Exposure time: 500 ms. Minimum-Maximum display range in ImageJ (Unsigned 16-bit range)=73–3500. G: Sagittal section of P21 brain injected at P4 with 4 μL of AAV9 (1 × 1013 vg/mL. Exposure time: 1000 ms. Minimum-Maximum display range in ImageJ (Unsigned 16-bit range)=73–2000. (H) Quantification of cortical and thalamic neuron labeling at P6, P9, and P14 after P1 (P6, n = 5; P9, n = 2; P14, n = 5; P45, n = 5) or P4 (P14, n = 2) sinus injections. Each data point in the plot represents an individual brain. Horizontal lines represent the mean, and vertical lines represent the standard deviation. C = cortex, T = thalamus. (I). Quantification of neuron labeling at P14 in different cortical and thalamic regions, in addition to hippocampus and striatum. M1 = motor cortex (n = 5), V1 = visual cortex (n = 4), S1 = somatosensory cortex (n = 3), A1 = auditory cortex (n = 2), RSA = retrosplenial cortex (n = 2), Piri = piriform cortex (n = 3), DLG = dorsolateral geniculate nucleus (n = 2), ADT = anterodorsal thalamic nucleus (n = 1), Hipp = hippocampus (n = 3), CPu = caudate and putamen (n = 2), SC = superior colliculus (n = 2). Confocal images showing GCaMP6s expression in mouse cortex and thalamus (J–W) at P14 after transverse sinus injection of 4 μL of 1 × 1013 vg/mL AAV9-syn-GCaMP6s at P1. Panels (J), (O), (T) show abundant GCaMP6s expression in cortex, thalamus, and hippocampus, and localization with both NeuN and DAPI (M,R,W). Scale bar = 20 μm. (N). Confocal image of GCaMP6s revealing dense and widespread expression across all cortical layers at P14. Scale bar is 40 μm.